Pipettes are devices used to dispense liquid volumes accurately. Typically, the volumes so dispensed are less than 10 ml. Pipettes are commonly used in clinical, environmental, pharmaceutical, and chemistry laboratories. Manual action and automated pipettes perform their function typically by means of actuation of a piston within a cylinder. As the piston is drawn into the cylinder, a volume of liquid is drawn out of a reservoir. When the piston is returned to its original position that volume of liquid is expelled into a container placed so as to receive it. The volume of liquid delivered is dependent on the mechanism actuating the piston, the swept area of the piston/cylinder combination, the stroke of the piston, the condition of the various components within the system, temperature and other factors depending on the design of the particular pipette.
In may applications of pipettes it is important to know what average volume of liquid is so delivered (accuracy of delivery), and how repeatably it is delivered (precision of delivery). In the event that the accuracy and precision of delivery does not meet specified standards, then the pipette must be adjusted, repaired, or replaced. The frequency with which a given pipette must be checked depends on the criticality of results obtained in using it, and may range from daily to yearly, depending on its usage.
One way of checking the calibration of a pipette is to dispense distilled water into a container and to weigh the water so dispensed. Disadvantages of using this "gravimetric" method include the requirement for a balance of suitably high accuracy and sensitivity, a weight set with which to calibrate it, and a suitable location with a high degree of isolation from vibration and air currents; evaporation of the water sample, leading to error in the results; and the non-portability of the balance, meaning that all pipettes must be brought to the balance for checking of calibration. For large automated pieces of pipetting equipment this requirement is difficult or impossible.
An alternate method of calibrating pipettes is to use the pipette to dispense samples of dye solution or other absorbing chemical species. The sample so dispensed may be diluted with a diluent so as to reduce the absorbance to be within a range readily measured by a photometer. By knowing the concentration and absorbance properties of the dye solution, the volume of diluent, and the absorbance of the resulting mixture, the delivery volume of the pipette may be readily calculated.
This method, called a "colorimetric" method, has particular advantage for small pipette volumes (e.g. less than 100 microliters), as the weights of such small volumes of water are difficult to determine with a high degree of accuracy. Although an improvement over the "gravimetric" methods described above, known "colorimatic" assays typically suffer from a non-linearity in dye absorbance, requiring a complicated correction calculation.